The Effect of Chlorothalonil on Immune System Function in Fish and OystersEPA Grant Number: U915013
Title: The Effect of Chlorothalonil on Immune System Function in Fish and Oysters
Investigators: Baier-Anderson, Caroline L.
Institution: University of Maryland
EPA Project Officer: Lee, Sonja
Project Period: January 1, 1996 through January 1, 1999
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1996) RFA Text | Recipients Lists
Research Category: Fellowship - Toxicology , Academic Fellowships , Health Effects
The objective purpose of this research project study is to characterize the effects of the fungicide chlorothalonil (TCIN) on the phagocytic cells of the immune system of the striped bass, Morone saxatilus, the mummichog, Fundulus heteroclitus, and the oyster, Crassostrea virginica.
1. In vivo exposure studies were employed to determine if TCIN alters tissue macrophage function. Fish (F. heteroclitus) were exposed to TCIN (0.1-10 ppb) for 48 hours. Following the exposure, livers and anterior kidneys were collected. Microsomal and cytosolic fractions were prepared from the livers; proteins from each fraction were separated by polyacrylamide gel electrophoresis, transferred to PVDF membranes, and probed for cytochrome P450 (microsomes) or nitric oxide synthase (cytosols) induction using Western blotting techniques. Macrophages from the anterior kidney were collected and assayed for immunostimulated reactive oxygen species (ROS) production. 2. In vitro exposure studies were used to characterize the direct effects of TCIN on macrophages (M. saxatilus) and hemocytes (C. virginica). Macrophage-enriched cell populations from M. saxatilus anterior kidney or from oyster hemocytes were collected, and aliquots of cells from individuals were exposed to a range of TCIN (10-500 ppb) for 20 hours. Some exposures included pre- or co-incubation with buthionine sulfoximine (BSO, to reduce cytosolic glutathione concentration) or dithiothreitolT, to protect sulfhydryl groups). Following the exposure, several assays were conducted to evaluate TCIN-induced toxicity, including cell viability, phagocytosis, and immunostimulated ROS production. Additionally, immunostimulated NADPH production was measured, and the thiobarbituric acid reactive substance (TBARS) assay for lipid peroxidation was performed.